PROJECT SUMMARY/ABSTRACT Cancer vaccines have recently emerged as a selective way to activate T cells against cancer. The treatments are based on peptides, called tumor antigens, derived from peptide sequences in tumors that are immunogenic. Another class of tumor antigens, called neoantigens, arise from mutations in tumors that have not been detected by the immune system. These cancer vaccines with tumor antigens or neoantigens can promote antigen-specific, rather than nonspecific, T-cell activation, by either generating new or amplifying existing immune responses against tumors. Despite the promising anti-cancer immune responses observed with these treatments, effective and selective activation of the immune system remains difficult. Several challenges have limited the translation of cancer vaccines into the clinic: (1) Sufficient uptake of the antigens by antigen presenting cells is difficult to achieve, which greatly influences whether the peptides are processed or presented to T cells. (2) Proteasomal processing of the peptides is difficult to predict, which can result in the formation of peptide fragments, other than the desired antigen, that neither bind to an HLA allele nor activate an immune response. Antigen delivery systems can play crucial roles in improving cancer vaccines. The delivery systems can perform two critical functions, which the absence of currently limits the efficacy of cancer vaccines: promote targeting to dendritic cells (DCs) and facilitate cytosolic delivery. This proposal describes the development of an anthrax delivery system for delivering tumor antigens. My overarching hypothesis is that the anthrax machinery is well suited for delivering tumor antigens, because it can efficiently perform protein translocation. In nature, the delivery system transports toxins into the cell cytosol that rapidly induce cell death. The main components are easily modified for transporting non-native cargo into cells, including therapeutic peptides, proteins, and even small molecules. This proposal will develop the anthrax delivery system with two new features: to target dendritic cells and to deliver tumor antigens. These features will be developed to enhance tumor antigen activity in vivo (Aim 1) and to shed light on antigen processing and presentation (Aim 2). The impact of this work will extend beyond developing an effective tool for tumor antigen delivery. It will also facilitate the identification and study of tumor antigens selective for T cell activation, which will ultimately lead to the development of better tumor antigens for cancer vaccines in the clinic and beyond. The training plan and environment permits the design and study of the anthrax delivery system with the Pentelute lab (MIT), Irvine lab (Koch Institute for Integrative Cancer Research at MIT), and Wu lab (Dana- Farber Cancer Institute). The proposed studies will be performed with the equipment and resources available in these labs, and with the facilities available at the Swanson Biotechnology Center at the Koch Institute.